High throughput genetic screening of lipid and cholesterol processing using fluorescent compounds

ABSTRACT

The present invention utilizes fluorescent lipids, particularly quenched phospholipid or cholesterol analogues, to facilitate screening for phenotypes representing perturbations of lipid processing; screening for genetic mutations that lead to disorders of phospholipid and/or cholesterol metabolism; and screening of compounds designed to treat disorders of phospholipid and/or cholesterol metabolism. The present invention is a mutant fish containing a metabolic defect affecting phospholipid and cholesterol processing. The mutant fish is useful for studying phospholipid and cholesterol metabolism and for screening compounds designed to treat disorders of phospholipid and/or cholesterol metabolism.

CONTINUING APPLICATION DATA

[0001] This application claims priority under 35 U.S.C. §120 based uponU.S.

[0002] Non Provisional Application No. 09/974,550 filed Oct. 10, 2001and upon U.S. Provisional Application No. 60/264,977 filed Jan. 30,2001.

GOVERNMENT RIGHTS TO THE INVENTION

[0003] The invention was made with government support under grant F32NS103265 awarded by the National Institute of Health. The government hascertain rights to the invention.

FIELD OF THE INVENTION

[0004] The present invention generally relates to the fields ofbiochemistry and pharmacology and to a mutant fish having a metabolicdefect affecting phospholipid and cholesterol processing, and moreparticularly to the use of a mutant fish labeled with fluorescent lipidsto screen for drugs and genetic alterations related to phospholipidand/or cholesterol metabolism and, more particularly, to the use of amutant zebrafish in conjunction with tagged or quenched lipids forstudying lipid metabolism in vivo,.

BACKGROUND OF THE INVENTION

[0005] Genetic analysis in zebrafish is a powerful approach foridentifying genes that direct vertebrate development (1-3). Since thecompletion of the large-scale chemical mutagenesis screens in 1997, thephenotypic and molecular characterizations of many mutations have beenreported (4-16). Analyses of mutations that affect early developmentalprocesses, such as the specification of the embryonic axes and germlayers, have been particularly rewarding (7, 10, 17-27). Recently,related work with mutations that affect organogenesis has led to therecognition that the zebrafish is an important model system forbiomedical research (28-31). Given the many aspects of organ physiologythat have been conserved during vertebrate evolution, genetic screeningto assay organ function in the optically transparent zebrafish is avaluable approach to understanding a variety of metabolic processes anddisorders in vertebrates.

[0006] By zebrafish chemical mutagenesis screening, nine recessivelethal mutations that perturb development of the digestive organs wereidentified (2, 31). Although the mutants were identified usingmorphological criteria, their phenotypic analysis suggests that in somecases the affected genes regulate developmental processes that arerelevant to digestive physiology and other aspects of vertebratemetabolism.

[0007] Through the analysis of these and other zebrafish mutants, thelimitations inherent to genetic screens that are based solely onmorphological criteria became apparent. First, not all organs arereadily distinguished in zebrafish larvae, and mutations that perturborgan morphology are often overlooked. Second, since it is difficult tovisualize specific cell populations within many larval organs, mutationsthat affect the development or function of these cells can be overlookedas well. Third, despite the transparency of the zebrafish larva, thefunction of few organs can be effectively assayed by visual inspectionalone.

[0008] For these reasons, it was concluded that, in most instances,morphology-based screens are best suited for the identification of genesthat regulate specification and patterning of embryonic structures. Bycontrast, screens designed to address biomedical concerns are mosteffective when they assay physiological processes directly.

[0009] Within the past few years, the discovery and analysis ofzebrafish mutants affecting organogenesis has confirmed an importantrole for the zebrafish in biomedical research. The ability to apply highthroughput genetic analyses to vertebrate organ physiology using thismodel system is unprecedented and will undoubtedly, over time, lead tothe discovery of many genes that regulate vertebrate organ developmentand physiology. Such zebrafish research will complement research inother vertebrate model systems.

[0010] By conducting a mutagenesis screen using fluorescent lipids, anundertaking not feasible with standard zebrafish screening strategies,the power of high throughput genetic analysis can be applied to lipidmetabolism. This has important implications for human diseases such as,but not limited to, cancer, inflammatory and cardiovascular diseases,and congenital and acquired diseases of the intestine and liver.

[0011] The fluorescent phospholipase A₂ (PLA₂) substrates described inthe present invention are the first prototypes in this class ofreagents. Although lipid metabolism in the digestive tract is complexand involves multiple organs the present invention discloses a method ofassaying this pathway since gall bladder fluorescence represents one ofthe last steps in lipid processing. Because they serve as reporters oflipid processing, the fluorescently-tagged reagents of the instantinvention provide a sensitive assay for a wide range of digestivedevelopmental and physiological processes including, but not limited to,swallowing; lipid digestion, absorption, and transport; esophagealsphincter function; intestinal motility; organogenesis of the mouth andpharynx, esophagus, intestine, liver, gallbladder and biliary system,and exocrine pancreas and ducts; and the cellular and molecular biologyof PLA₂ regulation, polarized transport, and secretion.

[0012] Given the shared features of lipid processing in mammals andteleosts (82, 105), zebrafish mutagenesis screens using lipid reporterscan be used to identify genes with functions relevant to human lipidmetabolism and disease. Moreover, since both mammals and teleostsmetabolize lipids in an analogous manner, the high throughput screensand fluorescent lipids disclosed in the instant invention can beemployed using a variety of vertebrate model systems, including but notlimited to, rodents, amphibia, and fish. The present invention involvesutilizing fluorescent lipids to screen for phenotypes representingperturbations of lipid processing; to screen for mutations of specificgenes that lead to disorders of phospholipid and/or cholesterolmetabolism; and to screen for compounds designed to treat disorders ofphospholipid and/or cholesterol metabolism, such as, but not limited tocancer, inflammatory and cardiovascular disease, and congenital andacquired diseases of the intestine and liver.

[0013] Because the precise mechanism of phospholipid and cholesterolprocessing is unknown at this time, there exists a need in the art formodels to study such metabolism. In order to study phospholipid andcholesterol metabolism, especially in vivo, one of the best approachesis to generate genetic mutant organisms, such as fish, that have ametabolic defect affecting phospholipid and cholesterol processing. Thisapproach allows the creation of new fish strains for physiological studyas well as tissues and cell lines for in vitro examinations. The presentinvention is further directed to a mutant fish having a metabolic defectaffecting phospholipid and/or cholesterol processing. The invention isuseful for the study of phospholipid and/or cholesterol metabolism and,hence, for the development of treatments of diseases or disordersrelated to phospholipid and/or cholesterol metabolism. In addition, thisinvention relates to isolated cells derived from these fish and to theuse of these cells in assays for assessing treatments of diseases ordisorders related to phospholipid and/or cholesterol metabolism.

[0014] Abbreviations

[0015] “PLA₂” means “phospholipase A₂”

[0016] “PLA₁” means “phospholipase A₁”

[0017] “PLB” means “phospholipase B”

[0018] “PLD” means “phospholipase D”

[0019] “PED6” means“N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-palmitoyl-2-BODIPY-FL-pentanoyl-sn-glycero-3-phosphoethanolamine”

[0020] “hpf” means “hour post-fertilization”

[0021] “dpf” means “day post-fertilization”

[0022] “FRET” means “fluorescence resonance energy transfer”

[0023] “PC” means “phosphatidylcholine”

[0024] “TLC” means “thin layer chromatography”

[0025] “cPLA₂” means “cytoplasmic PLA₂”

[0026] “sPLA₂” means “secretory PLA₂”

[0027] “COX” means “cyclooxygenase”

[0028] “APC” means “adenomatous polyposis coli”

[0029] “EP” means “early pressure”

[0030] “ENU” means “ethylnitrosourea”

[0031] “WT” means “wild-type”

[0032] “SLR” means “single locus rate”

[0033] “IVF” means “in vitro fertilization”

[0034] “BAC” means “bacterial artificial chromosome”

[0035] “PAC” means “P1-derived artificial chromosome”

[0036] “YAC” means “bacterial artificial chromosome”

[0037] “SSR” means “simple sequence report”

[0038] “CSGE” means “conformation sensitive gel electrophoresis”

[0039] “VLDL” means “very low density lipoprotein”

[0040] “EM” means “embryo medium”

[0041] “NBD cholesterol” means “nitrobenzoxadiazole cholesterol”

[0042] Definitions

[0043] As used herein, the term “agent” refers to any molecule,compound, or treatment that assists in the treatment of a disease ordisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism.

[0044] As used herein, the term “screening” or “to screen” refers to aprocess in which a large number of potentially useful agents areprocessed in the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1. Schematic of PED6, a quenched PLA₂ substrate. A. Thedinitrophenol on the phospholipid headgroup effectively quenches anyemission resulting from excitation at 505 nm of the BODIPY-labeled acylchain. B. When the BODIPY-labeled acyl chain is liberated by PLA₂mediated cleavage, the quencher is separated from the fluorophore andemission (515 nm) is observed. C. NDB-labeled cholesterol in which thefluorophore replaces the terminal segment of cholesterol's alkyl tail.

[0046]FIG. 2. Intestinal differentiation in 84 hpf larvae. A.Cross-section of posterior intestine. Desmin immunoreactivity ofintestinal smooth muscle (green) and enteric neurons as indicated by zn6immunoreactivity (red) demonstrate development of the entericneuromuscular system. B. Transmission Electromicrograph showing matureapical junctional complexes in enterocytes in the intestinal epithelium.Arrow points to desmosomes; A: Adherens junction. T: tight junction. C.Histochemical detection of enterocyte aminopeptidase activity (red);anterior intestinal cross section. D. Histological detection of gobletcell mucin (purple) in the posterior intestine. Also present at thisdevelopmental stage, immunoreactive pancreatic polypeptide inenteroendocrine cells.

[0047]FIG. 3. PED6: a fluorescent lipid reporter. A. Bright-field imageof a 5 dpf larva soaked in PED6 (3 ug/ml, 2 hr). B. Correspondingfluorescent image, with intestinal (arrow) and gall bladder (arrowhead)labeling. C. Larva soaked in BODIPY-C5-PC (0.2 ug/ml). In contrast to B,unquenched fluorescent lipid labels the pharynx (arrowhead), confirmingthat lipid is swallowed before gall bladder labeling (arrow).

[0048]FIG. 4. Rate of fluorescence after PED6 labeling. Larvae (n=5)were placed in medium containing PED6 (0.17 mg/ml) and tricaine. Imageswere captured at various times and fluorescence intensity was determinedin specific structures. Organ fluorescence intensity determined atspecific times was normalized to the observed intensity at 45 min. Dataare expressed as Mean±SEM.

[0049]FIG. 5. Lipid processing. A. and B. Atorvastatin (ATR) inhibitsprocessing of PED6 (A) but not of BODIPY-FL-C5 (Molecular Probes). B.Larvae were bathed in fluorophore (0.6 uM) in the presence or absence ofatorvastatin (Lipitor tablet suspension containing 1 mg/ml) (arrowhead,gall bladder). C. Mouse digestive organs. D. Gall bladder fluorescenceafter processing (t=30 min) of PED6 (1 ug), administered by gavage.Symbols: gb, ball bladder; d, common bile duct; lv, liver. Scale bars,1.0 mm (C and D), 200 um (other images).

[0050]FIG. 6. BODIPY FR-PC reveals both the substrate and PLA₂ cleavageproduct. Upon integration into cells, excitation at 505 nm results in anemission at 568 nm (orange) due to fluorescence resonance energytransfer (FRET). After cleavage by PLA₂ emission is observed only at 515nm (green).

[0051]FIG. 7. Fluorescence emission spectrum of mixed micelles of 0.05mol % BODIPY FR-PC in mixed-lipid vesicles. The fluorescence emissionspectrum of BODIPY FR-PC (0.5 uM in ethanol; excitation, 505 nm) showedpeaks at 514 nm and 568 nm with a ratio of 1.0 indicating FRET. Anexcitation scan (emission, 568 nm) showed peaks at 507 and 560 nm with aratio (507/560) of 1.2. Solid line: before addition of PLA₂. Dashedline: after addition of N. naja PLA₂. Vesicles were prepared bysonication of the dried lipids (from chloroform-methanol solution); 1.1μM BODIPY FR-PC (0.05 mol %), 0.1 mM dimyristoylphosphatidycholine (46mol %), 0.12 mM ditetradecylphospha-tidylmethanol (54 mol %), buffer (50mM Tris, pH 8, 100 mM NaCl, 1 mM CaCl₂); excitation, 505 nm.

[0052]FIG. 8. BODIPY FR-PC is effective in paramecium. Parameciumincubated (1 hr) in BODIPY FR-PC (2.5 ug/ml) results in labeling oflipid droplets (orange). Digestion of the lipid results in greenfluorescence. Excitation 505 nm, emission LP520.

[0053]FIG. 9. BODIPY FR-PC localizes to the intestinal epithelium. Fishwere incubated (1-4 hr) in BODIPY FR-PC (2.5 ug/ml). BODIPY FR-PCmetabolites are observed in gall bladder (green). Excitation 505 nm,emission LP520. A. Brightfield image. B. and C. Visualization of antigenpresenting enterocytes in segment II of the larval zebrafish intestineafter only 1 hr of labeling. Arrows mark segment II domain. D. 4 hrlabeling with BODIPY FR-PC results in FRET effect throughout intestine.

[0054]FIG. 10. Lipid processing in intestinal mutants. A and C. Brightfield images of 5 dpf mlt and pie larvae. B and D. Fluorescent imagescorresponding to A and C. Normal lipid processing of PED6 (0.3 ug/ml, 2hr) in mlt larva (arrowhead marks gallbladder). D and E. Abnormal lipidprocessing in PED6 labeled pie and slj larvae; fluorescence is presentin the intestinal lumen (white arrowhead) and reduced in the gallbladder(red arrowhead).

[0055]FIG. 11. Phospholipid processing and transport. A. Followinguptake, PED6 is cleaved by PLA₂ liberating a labeled fatty acid. Thefate and transport of this fatty acid remains unknown. B. TLC offluorescent lipid standards. PED6 (black because it is quenched), D3803(C5-BODIPY PC), and C5-fatty acid are easily resolved using a twosolvent system silica gel plates (Whatman, LK5D). Solvent 1 (toluene,ether, ethanol, acetic acid; 25/15/2/0.2); Solvent 2 (chloroform,methanol, acetic acid, water; 25/15/4/2).

[0056]FIG. 12. Red BODIPY-PC given by gavage labels the gall bladder.Adult fish was anesthetized in tricaine, injected with 80 ug of D3806 (aBODIPY PC 582/593 nm), allowed to recover for 1 hr and dissected on ice.

[0057]FIG. 13. Larval zebrafish (5 dpf) labeled with NDBcholesterol—derivative labels gall bladder within 30 minutes ofingestion (3 ug/ml, solubilized with fish bile).

[0058]FIG. 14. fat free larvae lack gall bladder fluorescence. A.Bright-field images of wild-type (WT) and mutant 5 dpf larvae. B.Corresponding fluorescent image of the gall bladder after PED6 labeling(0.3 ug/ml) (arrowheads). C. Biliary secretion of phenol red in fat freelarvae. Mutant larvae identified by PED6 labeling were injected withphenol red (arrowhead, gall bladder). D. Reduced lipid processing in fatfree mutants. TLC of the pooled lipid fraction from eight larvae isshown after labeling with BODIPY C5-PC (12 hours, 0.16 ug/ml); redarrowhead denotes BODIPY-cholesteryl ester, black arrowhead indicatesBODIPY C5-PC (lane 1, WT; lane 2, fat free; lane 3, endogenousfluorescent lipids present in unlabeled WT larvae). TLC: solvent 1(chloroform:methanol:water 35/7/0.7); solvent 2 (ethylether:bensene:ethanol:acetic acid; 40/50/2/0.2). E. fat free larvae failto accumulate22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol(NBD-cholesterol) (2 hrs, 3 ug/ml, solubilized with fish bile). Scalebars, 200 μm.

DETAILED DESCRIPTION

[0059] The present invention relates to a mutagenesis screen to identifygenes that regulate lipid metabolism using fluorescently-tagged orquenched lipids such as cholesterol or lipids that are substrates forphospholipases such as PLA₂ and to a fish having a metabolic defectaffecting phospholipid and cholesterol processing, which is identifiedusing a mutagenesis screen. For example, cleavage of quenchedphospholipid substrates by PLA₂ results in an increase in fluorescenceintensity or alters the spectral properties of fluorescent emission thusallowing lipid metabolism to be followed in vivo. In one embodiment ofthe instant invention, optically transparent zebrafish larvae exposed tothe fluorescently-tagged or quenched lipids display intense gallbladderfluorescence, which reflects lipid cleavage by intestinal PLA₂ andsubsequent transport of the fluorescent cleavage products through thehepatobiliary system.

[0060] The instant invention presents evidence demonstrating that in thecontext of a mutagenesis screen, fluorescent PLA₂ substrates andfluorescently-tagged cholesterol or other lipids have the potential toidentify genes that affect many aspects of lipid metabolism.Consequently, when used in the context of a genetic screen thesereagents provide a high throughput readout of digestive physiology thatcannot be assessed using standard screening strategies. Given currentunderstanding of the pathway of lipid processing in zebrafish,specifically its shared features with lipid processing in mammals, thepresent invention has relevance for biomedical research related to,among other things, cancer, inflammatory and cardiovascular diseases,and congenital and acquired diseases of the intestine and liver.

[0061] Fluorescent Reagents to Assess Organ Physiology in vivo

[0062] The fluorescent lipids of the instant invention allow assaying ofphysiological processes. The reagents are fluorescent analogues ofcompounds that could serve as modifiable substrates in importantmetabolic and signaling pathways. The reagents of the instant inventionwere constructed by covalently linking fluorescent moieties to sitesadjoining the cleavage site of phospholipids. Dye-dye or dye-quencherinteractions modify fluorescence without impeding enzyme-substrateinteraction (106). PLA₂ cleavage results in immediate unquenching anddetectable fluorescence. Quenched phospholipids[N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-palmitoyl-2-BODIPY-FL-pentanoyl-sn-glycero-3-phosphoethanolamine(PED6)] allow subcellular visualization of PLA₂ activity and revealorgan-specific activity (36). The evidence presented in the instantinvention demonstrates that zebrafish larvae 5 days post-fertilization(dpf) bathed in PED6 show intense gall bladder fluorescence and, shortlythereafter, intestinal luminal fluorescence. Substrate modificationallows localization of enzymatic activity by altering the emissionspectrum of the fluorescent compounds. When used in the context of agenetic screen these fluorescent lipids provide a high throughputreadout of organ function.

[0063] The reagents of the instant invention facilitated the developmentof genetic screens that are more sensitive than the whole-mount in situand antibody based screening protocols now used to assay geneexpression. The fluorescent reagents are simpler to use since they canbe administered to and assayed in a wide range of organisms, including,but not limited to rodents and teleosts, and they offer the opportunityto screen for hypomorphic mutations that alter gene function but do notaffect levels of gene expression. By providing a visual assay ofmetabolic processes, these reagents can be used to identify mutationsthat affect more than just the single gene responsible for substratemodification. Visualization of the fluorescent signal also is dependentupon the delivery and uptake of the substrate as well as the storage,metabolism or secretion of its modified metabolites.

[0064] Fluorescent PLA₂ Substrates

[0065] PLA₂s were chosen as the “target” enzymes to assay because of theimportant role these enzymes play in the generation of lipid signalingmolecules (32-34). PLA₂s are a large family of enzymes that can becategorized according to their cellular distribution, molecular weight,and calcium dependence (33). Some PLA₂s also exhibit a preference forphospholipids with arachadonyl sn2 acyl side chains (34, 37, 38). Giventhe wide range of processes the PLA₂ family of enzymes is known orthought to regulate, the advantage of the fluorescent lipids asscreening reagents is significant.

[0066] cPLA₂. For example, one PLA₂ gene family member, cytoplasmic PLA₂(cPLA₂), regulates eicosanoid production. Eicosanoids are ubiquitoussignaling molecules generated by the biochemical modification ofarachidonic acid, the principal fatty acid liberated from membranephospholipids by cPLA₂s (42-44). cPLA₂s have a strong preference forarachidonyl phospholipids and cPLA₂ cleavage of these phospholipids isknown to be the rate-limiting step in eicosanoid synthesis (39, 41,44-47). In comparison, secretory PLA₂s (sPLA₂s) exhibit little substratepreference and unlike cPLA₂, the arachidonic acid generated throughsPLA₂ activity is not directly coupled to eicosanoid production (37,46).

[0067] The two major classes of eicosanoids produced by vertebrate cellsare leukotrienes and prostaglandins (48, 54). Prostaglandins are a largefamily of signaling molecules that are synthesized through the action ofcyclooxygenases (COX) and prostaglandin-isomerases on PLA₂ generatedarachidonic acid (48). Prostaglandins are especially important invertebrate physiology as they regulate a myriad of physiologicalprocesses, including hemostasis, cell proliferation, fertility, andinflammation (48-50). The importance of prostaglandin in humans isunderscored by the widespread use of COX inhibitors (e.g., aspirin) asmedicinal agents.

[0068] Given the focus of this invention, there is special interest inintestinal prostaglandins. Intestinal prostaglandins play an importantrole in regulating mucosal blood flow (51-54), and inhibition ofprostaglandin synthesis by COX inhibitors, such as aspirin, isassociated with the development of mucosal ulcerations (55). There isalso strong evidence that intestinal prostaglandins regulate epithelialcell proliferation (56-58). COX inhibitors are known to have achemopreventitive effect on several human digestive cancers, and COX-2has been identified as a genetic modifier of APC, an important coloncancer gene.

[0069] Because arachidonic acid release is the rate-limiting step ineicosanoid production, characterization of the cell specific regulationof cPLA₂s is important (33, 34). Although the genes in some of thesignaling pathways that activate cPLA₂ have been identified, thischaracterization is far from complete. A screen utilizing fluorescentlipid substrates will allow identification of mutations that perturblipid processing and alter cPLA₂ activity.

[0070] sPLA₂. Another PLA₂ gene family member, sPLA₂, plays a role ininflammation, host defenses, digestion, and cell proliferation (43, 70,71). In comparison to cPLA₂s, sPLA₂s show little preference forarachidonic acid over other phospholipids sn2 acyl side chains (34).

[0071] There is a large body of experimental and clinical evidencesupporting the role of sPLA₂s in inflammation. First, serum levels ofsPLA₂ are increased in inflammatory conditions such as RheumatoidArthritis, Crohn's Disease, endotoxic shock, and atherosclerosis (32,72-76). Second, sPLA₂ cleavage of arachidonyl phospholipids has beenshown to augment cPLA₂ mediated eicosanoid production (46, 71, 77).Third, independent of its enzymatic activity, sPLA₂ has been shown tobind to a specific cellular receptor that plays a role in endotoxicshock (78) and other aspects of the inflammatory response (43, 79, 80).Like cPLA₂, the regulation of inflammatory sPLA₂s (Groups II and V) isan important area of biomedical research.

[0072] Group IIA sPLA₂ recently has been shown to play a role in coloncancer tumorigenesis through its genetic interaction with APC in the minmouse, a colon cancer animal model (66-68). High levels of this gene areexpressed in intestinal Paneth cells where it is also believed tofunction as an antimicrobial agent because of its ability to digestbacterial cell wall phospholipids (38, 76, 81). The precise mechanism ofsPLA₂'s mitogenic effect is unclear. Since COX activity also has beenshown to modify colon tumorigenesis in the min mouse, sPLA₂ may beacting via prostaglandins (69). This hypothesis is counter-intuitive,however, because in the min mouse, loss of sPLA₂ function is associatedwith a tumor promoting effect. An alternate hypothesis suggests that inthe colon, sPLA₂ also may function as a cell-signaling molecule,independent of its enzymatic activity, through its interaction with thesPLA₂ receptor.

[0073] The role of sPLA₂s in the digestion and absorption of dietaryphospholipids has been studied in many vertebrate species, includingteleosts (82). Pancreatic sPLA₂ is the prototypical low molecular weightsPLA₂—its proenzyme is secreted into the intestinal lumen where it isactivated and cleaves dietary phospholipids that have been modified bybile salts (83). PLA₂ activity also is present in the intestinal brushborder and undoubtedly contributes to the digestion of dietaryphospholipids (84, 85). Intestinal PLA₂ activity is present in the formof PLB, an enzyme that can function as a high molecular weight, calciumindependent PLA₂ as well as a lysophospholipase (86-89). Regulation oflipid absorption and transport is an important area of biomedicalresearch that has implications for metabolic, inflammatory, andcardiovascular diseases.

[0074] Phospholipid Metabolism

[0075] In fish, like mammals, dietary phospholipid is cleaved byintestinal and pancreatic PLA₂s to form free fatty acid, lysolipid, andphosphoglycerol (82). These molecules are absorbed by enterocytes,presumably by simple diffusion (lysolipid, phosphoglycerol) and receptormediated processes (fatty acid) (90). Within the enterocyte, the freefatty acids are processed according to their size: long carbon chainfatty acids are re-esterified to form triglycerides and phospholipids,packaged into lipoprotein particles (chylomicrons or VLDL) and enter thegeneral circulation via the lymphatics; shorter chain fatty acidspresumably can enter the circulation directly via the portal vein(90-92). Lipoprotein bound phospholipids enter cells in the periphery bybinding to specific receptors or via endocytosis (90, 91).

[0076] In comparison to the fatty acids, the fate of the phospholipidderived lysolipid and phosphoglycerol cleavage products are lesscertain. In mammals, they are largely re-esterified to formphospholipids, which then are incorporated into lipoproteins andabsorbed via the lymphatics, like triglycerides (90-92). Vertebratelipid absorption is further complicated by the existence of otherintestinal phospholipases (PLD, PLA₁) that cleave the polar head groupor snl fatty acid from lysolipids and phospholipids. This raises thepossibility that phospholipids undergo extensive re-arrangement withinthe intestinal epithelium prior to absorption. In fish, there isexperimental evidence supporting this observation (82).

[0077] Materials, Methods, and Results

[0078] Zebrafish

[0079] Zebrafish, Danio reno, were housed in a separate facilityconsisting of approximately 500 tanks of varying sizes (1 liter, 3.75liter, and 9 liter). Environmental conditions were carefully monitoredfor disease prevention and to maintain fish in perpetual breedingcondition. Male and female fish were reared at a density of no more than8 fish per liter at a constant temperature and light cycle (27-29° C.with the light/dark cycle kept at 14/10 hours) in pretreated water(heated, charcoal-filtered and UV-sterilized). Fish were fed twice dailywith a variety of dried and live foods.

[0080] Zebrafish provide a relatively simple model system for morecomplex vertebrates, such as humans. They are small in size, easy tomaintain and breed, and produce large numbers of progeny on a dailybasis. Their embryos develop rapidly and are optically clear, permittingdirect observation of the developing digestive system. Beingvertebrates, zebrafish contain orthologues for almost all human genes.The species also is amenable to genetic methods so that one can screenfor mutations that disrupt organ function or development. It ispossible, therefore, to identify genes important for intestinaldevelopment and function by examining fish that carry random mutations.In addition, many techniques have been worked out for manipulatingzebrafish, including in vitro fertilization, production of haploids andparthenogenic diploid embryos, mutagenesis, cell lineage and celltransplantation.

[0081] Generation of fluorescent PLA substrates to assay lipidprocessing

[0082] In the instant invention, a family of fluorescent phospholipidswas generated with such phospholipids serving as substrates for PLA₂. Aspreviously noted, characterization of the genetic regulation of PLA₂ isan active area of biomedical research (67, 68), and the reagentsdescribed were developed to function as in vivo biosensors of PLA₂activity that can be assayed using microscopy or simple biochemicaltechniques. When administered to zebrafish embryos and larvae, thesereagents provide a rapid readout of a wide range of developmental andphysiological processes that are amenable to high throughput geneticanalyses. The fluorescent lipids described in this proposal are quenchedfluorescent phosphatidylcholine analogues and NBD-labeled cholesterol(FIG. 1). The phospholipid reagents differ in both their fluorescentemission and their specificity for different PLA₂ isoforms (93, 94).Cleavage of these phospholipids by PLA₂ generates a fluorescent fattyacid or lysolipid that can be used to localize and quantify PLA₂activity in live fish (36, 94). It has been demonstrated that there isutility of these agents for revealing localized PLA₂ activity indeveloping zebrafish embryos (94).

[0083] To determine whether these reagents could be used to study organspecific PLA₂ activity, the quenched fluorescent lipids wereadministered to zebrafish larvae at 5 dpf, a developmental stage whenthe major larval organ systems function (FIG. 2). 5 dpf larvae soaked inPED6 uniformly developed intense gallbladder fluorescence 15-20 minutesafter ingesting the lipid (FIG. 3). Based upon established mechanisms ofvertebrate lipid processing, it was theorized that the larvalgallbladder fluorescence reflected ingestion of the lipid reagentsfollowed by intestinal PLA₂ cleavage, intestinal absorption, transportto the liver and biliary excretion of the fluorescent cleavage product.

[0084] This hypothesis was tested with three experiments. First, toestablish that PED6 is swallowed by the larvae, an unquenchedfluorescent lipid was administered to 5 dpf larvae, and the appearanceof labeled lipid in the pharynx and intestinal lumen before thegallbladder was noted (FIGS. 3C & 4). Second, that gallbladderfluorescence reflects hepatobiliary transport of the fluorescentcleavage products was shown by demonstrating the absence of PLA₂activity in dissected adult gallbladders with and without bile (4785±626when full of bile vs. 7421±2043 when empty, arbitrary fluorescence units±SEM, n=3). Dissected adult gall bladders were lysed in embryo medium(EM) (30 ul) containing BODIPY-FL-C₅-PC (0.1 ug) to release bile. PLA₂activity was determined as described (1). Measurements were comparedwith activity of bile-depleted gallbladders. Third, this finding wasconfirmed by demonstrating the early appearance of fluorescent cleavageproducts in the liver of larvae exposed to PED6 compared with thegallbladder (FIG. 4). Larvae were labeled with PED6 (0.3 ug/ml) in EM,anesthetized (tricaine, 170 ug/ml), and placed in depression slides.Fluorescent images were captured over 1 hr using a Zeiss Axiocam 2mounted on a Leica MZFL-III. Because PLA₂ activity was not detected inbile and fluorescent PED6 metabolites underwent rapid hepatobiliarytransport, labeling the liver before the gall bladder, PED6 must becleaved within the intestine.

[0085] Moreover, PED6 labeling of the gall bladder was completelyblocked by atorvastatin (Lipitor, Parke-Davis) (FIG. 5A), a potentinhibitor of cholesterol synthesis in humans (108). Because the additionof exogenous bile reversed this effect [Bile was obtained from freshlykilled tilapia (Orechromis mossambicus) and extracted with three volumesof methanol:chloroform (1:2). The aqueous fraction was recovered,reduced to one volume under nitrogen, and added to EM (20 ul/ml)], andatorvastatin failed to inhibit processing of a water-soluble short-chainfatty acid (BODIPY-FL-C5, Molecular Probes Inc.) (105) (FIG. 5B), thedata demonstrate that atorvastatin blocks the synthesis ofcholesterol-derived biliary emulsifiers needed for dietary lipidabsorption (109). These results, coupled with the rapid appearance ofbiliary fluorescence in mice fed PED6 (FIG. 5D), demonstrate that inzebrafish larvae, lipids are processed in a similar, if not identical,manner as other vertebrates and that gallbladder fluorescence reflectsintestinal absorption and hepatic excretion of the lipid reagents andits metabolites.

[0086] BODIPY FR-PC, a reporter of both substrate and cleavage product

[0087] To further demonstrate that PED6 and other fluorescent lipids areprocessed by intestinal lipases prior to transport to the liver andgallbladder (as compared to being absorbed from the intestinal lumen,transported to the liver, and first cleaved by hepatic PLA₂ activityprior to biliary excretion) a new fluorescent lipid was developed (FIG.6). BODIPY FR-PC (FIG. 6) is a substrate that can be used to determinethe site of PLA₂ activity more precisely because it emits distinctfluorescent profiles before and after cleavage. This phospholipidcontains two dyes that interact via fluorescence resonance energytransfer (FRET). Substrate and cleavage product possess unique spectralsignatures, allowing their tissue distribution to be distinguished byfluorescence microscopy. Excitation (505 nm) of micelle-incorporatedBODIPY FR-PC produces an orange emission (568 nm) (FIG. 7) from the sn2fluorophore via the FRET effect (18). Cleavage of the substrate purifiedby PLA₂, however, releases the sn2 acyl chain and abolishes FRET (FIG.7). Excitation of the resultant lysolipid produces a green emission (515nm). The fluorescence emission spectrum of BODIPY FR-PC was determinedusing mixed micelles of 0.05 mol % BODIPY FR-PC in dimyristoylphosphatidylcholine (46 mol %) and ditetradecylphosphatidylmethanol (54mol %) prepared in buffer (50 mM Tris, pH 8, 100 mM NaCl, 1 mM CaCl₂) bysonication of the dried lipids.

[0088] Having established that BODIPY FR-PC behaves as anticipated in invitro assays, its behavior in vivo was analyzed by exposing paramecia tothe lipid for 1 hr, followed by excitation at 505 nm. As expected, FRETemission (orange) was observed from lipid droplets within individualparamecium soon after exposure, followed by the gradual appearance ofBODIPY emission at 515 nm (green fluorescence) indicative of substratecleavage (FIG. 8).

[0089] To further demonstrate the behavior of BODIPY FR-PC in vivo, 5dpf zebrafish larvae were bathed in BODIPY FR-PC. Within 1 hr, larvaeexposed to BODIPY FR-PC showed a bright green fluorescent gallbladderand orange fluorescence within the apical cytoplasm of posteriorintestinal cells (FIG. 9C). These cells are known to absorb luminalmacromolecules through pinocytosis (107). Orange fluorescence indicativeof uncleaved substrate also was observed after 4 hr of incubation in theanterior intestine epithelium, the site of lipid absorption in fish (82,95). FRET emission was never detected in the liver even after prolongedincubation (FIG. 9D), implying that gallbladder fluorescence followingingestion of labeled lipids is due to cleavage by intestinal PLA₂s.

[0090] The evidence of the instant invention is consistent with thepredicted pathway of lipid processing observed in mammals sinceuncleaved substrate (orange) was seen only in the intestinal epithelium.Over time, continuous exposure to BODIPY FR-PC either overwhelmsintestinal lipase activity or leads to the integration of BODIPY FR-PCinto the cellular phospholipid pool. Regardless, the data of the presentinvention provide further evidence that in zebrafish, as in mammals,lipids are absorbed and modified in the intestine prior to transport tothe liver.

[0091] Lipid processing in zebrafish intestinal mutants

[0092] To determine the utility of the fluorescent lipids as reagents ina genetic screen, PED6 was administered to mutants known to perturbintestinal morphology (31), and the pattern and timing of gallbladderfluorescence was examined (FIG. 10). These mutations cause embryoniclethality and each of the affected genes regulate intestinal developmentin a region-specific manner. The mutations slim jim (slj) and piebald(pie) each cause degeneration of anterior intestinal epithelium andexocrine pancreas, whereas meltdown (mlt) results in cystic expansion ofthe posterior intestine. As shown in FIGS. 10B and 10C, mlt mutantsretain PED6 processing in the anterior intestine and show normal levelsof gall bladder fluorescence. In contrast, pie (FIGS. 10D & 10E) and slj(FIG. 10F) mutants display greatly reduced gall bladder labeling.Although slj and pie have similar histological phenotypes, gall bladderfluorescence was absent in pie larvae but visible in slj larvae, albeitat a reduced level compared with wild-type or mlt larvae (FIGS. 10B &10C). Hence, fluorescent lipids can be used to identify mutants withabnormal digestive organ morphology.

[0093] Screening for mutations that perturb lipid processing usingfluorescent

[0094] PLA 2 substrates

[0095] Lipid reporters also are effective tools for identifyingmutations that perturb lipid metabolism without causing obviousmorphological defects. In a pilot screen, larval progeny of individualF2 families derived from an ENU mutagenesis protocol were bathed in PED6and screened for digestive organ morphology and phospholipid processing.PED6 fluorescent metabolites dramatically enhanced digestive organstructure, facilitating scoring of gallbladder development, intestinalfolding, and bile duct morphology. From 190 genomes screened, twomutations were identified and confirmed in the subsequent generation.These mutations were recovered based on their pattern of gallbladderfluorescence, not morphological criteria, thereby supporting the use ofthe fluorescent lipids of the instant invention in large-scalemutagenesis screens.

[0096] Lipid processing was examined further in the identified mutantsusing a fluorescent cholesterol analog (FIG. 1).

EXAMPLE 1

[0097] Characterize the Biochemical Pathways Responsible for theMetabolism of the Fluorescent PLA₂ Substrates in Zebrafish

[0098] To characterize, biochemically, the identity of fluorescentcompounds present in the bile of zebrafish following exposure tofluorescent PLA₂ substrates and other lipid molecules, zebrafish wereexposed to a panel of fluorescent phospholipids and free fatty acidsthat differed in acyl chain length and fluorophore position. Theexperiments enabled the determination of the phenotypic significance ofrecovered mutants by identifying shared pathways of lipid metabolism inzebrafish and mammals.

[0099] Identity of the Fluorescent Lipid in the Bile of Fish Labeledwith PED6

[0100] To determine whether the fluorescence in the bile is due to areacylated phospholipid (lacks the DNP quencher on the head group) orexists as a free BODIPY-labeled fatty acid, adult fish were labeled withPED6 by gavage. Adult fish were used because of the small amount oflipid in larvae bile. Under a fluorescence stereomicroscope, the gallbladders were easily identified by their intense fluorescence. The gallbladders were removed at various times post-gavage and placed in coldmethanol, sonicated and extracted (97).

[0101] The organic fraction was then subjected to TLC analysis asdescribed to determine whether the fluorescent sn2 acyl chain wasre-esterified prior to biliary excretion (FIG. 11A & 11B) (36, 98). Thisprocedure was performed on five fish using D3806 (a red BODIPY PC,582/593) (FIG. 12).

[0102] Identity of the Fluorescent Lipids in the Bile of Fish Labeledwith BODIPY- C5, C12, C16 Fatty Acids

[0103] To ascertain whether short chain fatty acids are transported viathe portal system, adult zebrafish are injected with C16, C12 and C5BODIPY-labeled fatty acids and then the fluorescent bile characterizedbiochemically using TLC. Based on work in other vertebrates, onlyshort-chain fluorescent fatty acids can pass directly into the bile,consistent with transport via the portal vein, following injection ofC16 BODIPY fatty acid (82, 99). The profile and kinetics of labeling arevery different with the shorter chain analogues.

[0104] Labeling of 5 dpf Larvae with BODIPY Labeled Fatty Acids andPhospholipids

[0105] To extend the findings from the adult bile assays to youngerfish, the rate of gallbladder fluorescence in zebrafish larvae exposegdto BODIPY-labeled fatty acids and phospholipids with different chainlengths is compared. Larvae are soaked in the identical BODIPY fattyacids injected into the adults, and the rate of gall bladderfluorescence is assayed. The labeling rate is determined by placing afish in water containing the fluorescent substrate and capturing adigital image of a lateral view (Axiovision, Zeiss and ImageQuant,Molecular Dynamics Inc.). The gall bladder fluorescence is quantified atvarious time intervals to compute the rate of change over time. Delayedappearance of gallbladder fluorescence after exposure to long chainfatty acids and phospholipids lends further support to the hypothesisthat acyl chain length plays a role in lipid transport in zebrafish.

[0106] PLA₂ Isoform Mediation of PED6 Processing

[0107] It is well established that cPLA₂ and some iPLA₂ isoforms exhibitacyl chain specificity and favor phospholipids that contain arachidonicacid, the precursor of eicosanoids (41, 44). Despite the fact that thefluorescent phospholipids do not contain arachidonic acid, the placementof the BODIPY moiety on the sn2 position results in significant cleavageby cPLA₂ (36, 94). The ability of the BODIPY moiety to disrupt themembrane and its hydrophobicity are both properties similar toarachidonic acid that enable cleavage by cPLA₂ (100). Secreted PLA₂isoforms exhibit no acyl chain specificity (34). Fluorescentphospholipids that contain a saturated acyl chain on the sn2 positionare fine sPLA₂ substrates but poor cPLA₂ ones.

[0108] To identify which PLA₂ isoforms mediate the appearance offluorescence in the gall bladder, the rate of gall bladder fluorescenceis compared in larvae exposed to two types of substrates. Larvae arelabeled with two phospholipid substrates that differ in theirspecificity for cPLA₂: a PL with an sn1 BODIPY and an sn2 saturated acylchain—a poor PLA₂ substrate, or BODIPY FR-PC—a good cPLA₂ substrate. Iflittle difference is observed in the rates of gall bladder fluorescenceusing the two substrates, then the critical lipase important for theprocessing of these lipids is a secreted PLA₂ isoform, a promiscuousenzyme that exhibits little acyl chain preference (96). BODIPY FR-PC isused because no other available fluorescent phospholipid contains sn2arachidonic acid.

[0109] Taken together, the experiments clarify the relevant PLA₂isoforms, the route(s) of phospholipid/fatty acid transport, and allowrefinement of the screening reagents (e.g. by preparing cocktails offluorophores to target specific transport processes).

EXAMPLE 2

[0110] A Mutagenesis Screen to Identify Genes that Regulate LipidProcessing in Zebrafish

[0111] In the instant invention a large-scale genetic screen for genesthat regulate lipid processing in zebrafish using fluorescent PLA₂substrates is disclosed. This screen leads to the recovery of mutationsthat regulate a wide range of developmental and physiological processes.As outlined, the screen identifies mutations that either resemble or areallelic to previously described intestinal mutations. Given the largenumber of steps required for processing of the fluorescent PLA₂substrates, however, this screen leads to the recovery of genes thatcould not be identified using standard screening strategies. Theseinclude, but are not limited to, genes that directly regulate PLA₂;genes responsible for the development of the liver, biliary system, andthe intestinal vasculature and lymphatics; and genes that play a directrole in lipid metabolism and transport.

[0112] The mutagenesis and screening strategies of the instant inventionincorporate several well established methodologies. First, adult malefish are mutagenized with ENU using established dosing schedules. Thereexist a number of established dosing schedules (e.g., 110, 111). Second,the F1 progeny of the mutagenized GO fish will be bred to homozygosityusing a classical 3 generation protocol (F3 screen), as well asparthenogenetically, using the early-pressure (EP) technique (103).Progeny refers to any and all future generations derived or descendingfrom a particular organism, whether the organism is heterozygous orhomozygous for the mutation. Progeny of any successive generation areincluded herein, such that the progeny, the F1, F2, F3 generations, andso on indefinitely are included in this definition. Zebrafish larvaegenerated in this fashion are soaked in the BODIPY FR-PC, PED6, and /orNBD cholesterol (FIG. 13) lipid reagents at 5 dpf and then screened fordefects in lipid processing based upon the timing and pattern offluorescence in the intestine, liver and gallbladder. Parallel F3 and EPscreens are employed because they offer the best opportunity to maximizethe use of resources. Although EP screens are in many ways lessefficient than classical F3 screens, they are suited to moderate sizedfish facilities and require less manpower because of the reduced numberof matings required.

[0113] ENU Mutagenesis

[0114] For the EP screen, adult male fish from the ‘*AB’ zebrafish line(this line of fish carries the fewest number of haploinsufficient orlethal mutations of common lab strains) are exposed to ENU (3.5 mM) in asecluded fume hood for 30 minutes on 3 consecutive days. After takingthe necessary precautions to remove adherent ENU, the fish are returnedto the main fish facility and outcrossed to WT fish from the Wik geneticbackground 3 weeks after mutagenesis, and the F1 progeny are raised tosexual maturity. To improve the likelihood that the *AB mutagenizedmales do not carry significant mutations in their genetic background,mutagenized males are outcrossed only if 5 of their female siblingsproduce large clutches of viable EP embryos that survive to 5 dpf. Theefficacy of ENU mutagenesis is determined prior to outcrossing bycalculating the specific locus rate for new mutations at the alb, spa,and bra loci.

[0115] One week after ENU exposure, the mutagenized GO males are matedto double-mutant “tester” female carriers of the alb, spa, and bramutations and their 32 hpf progeny are analyzed for clones of alb/alb,spa/spa, and bra/bra mutant pigmented cells. Based upon published SLR'sfor these loci using our ENU dosing schedule there is a need to analyzeapproximately 1500-2000 progeny from each mutagenized GO fish toaccurately determine the efficacy of mutagenesis.

[0116] Screening for Mutations that Perturb lipid Processing

[0117] For screening, 5 dpf larvae derived from pair-wise matings of F2families or EP treatment of F1 females are soaked fluorescent lipids for1-10 hrs and analyzed for perturbation of lipid processing using afluorescent stereo-microscope (Leica MZ FLIII). The BODIPY FR-PC andPED6 reagents used in the screen are purified via TLC, resuspended inethanol/DMSO and tested in vivo using WT 5 dpf larvae prior toscreening. The F3 5 dpf larvae for screening are derived from pair-wisematings of fish from F2 families, while 5 dpf larvae producedparthenogenetically are produced using standard EP protocols: briefly,eggs are collected from anesthetized F1 females (tricaine) and exposedto UV irradiated sperm following standard in vitro fertilization (IVF)protocols. Immediately after IVF (1.4 min.), the fertilized eggs areexposed to 13,000 psi in a French Press for 4-6 min, then slowlyreturned to ambient atmospheric pressure and allowed to develop until 5dpf (103).

[0118] Putative mutations that alter the pattern or rate of accumulationof lipid fluorescence in the digestive organs, as well as those thatproduce specific alterations in larval morphology, are recovered andoutcrossed for future analysis. Mutations that result in a generalizeddelay in larval development are not analyzed given their high frequencyof recovery in prior chemical mutagenesis screens. For the F3 screen, noless than 20, 5 dpf larvae are analyzed from ≧6 crosses per F2 family.Using this strategy, excluding lethal effects of haploinsufficiency inheavily mutagenized genomes, the theoretical chance of recovering amutation present in the genome of an F2 family is ≧89%. For the EPscreen, all of the viable progeny derived from each F1 female areanalyzed. In contrast to the F3 screen, the statistical likelihood ofrecovering mutations in the genome of F1 females treated with EP is notpredictable given the tendency to recover smaller viable clutches withthis technique, and the potential for meiotic recombination to alterMendelian inheritance patterns. Only those heritable defects that can berecovered in statistically significant Mendelian ratios when outcrossed,are considered mutations.

[0119] The data and analysis of known intestinal mutants suggest thatlipid processing mutations are not rare mutations. A total ofapproximately 750-2000 mutanized genomes are screened.

Example 3

[0120] Determination of the Physiological Significance and MolecularNature of Recovered Mutations

[0121] The instant invention discloses methods for characterizing, bothphenotypically and molecularly, the generated mutations. Phenotypicanalysis is approached first. Mutations that affect a wide range ofdevelopmental and physiological processes are recovered in the screen.Careful phenotypic analysis of these mutants is important for severalreasons. First, it allows quantitation of the range and frequency ofphenotypes actually recovered using the fluorescent lipid reagents.Second, it allows the performance of comprehensive complementationanalyses so that hypomorphic alleles of interesting mutations are notoverlooked. Third, it allow the selection of those mutations worthy ofmolecular analysis now versus mutations that may be of more immediateinterest to other members of the zebrafish scientific community. Therationale for the molecular analysis of zebrafish mutations is wellknown to those of skill in the art. Recognition of the importance ofphenotypic analyses of mutant phenotypes prior to molecular analyses isrelevant given the large amount of work that is often required toidentify the responsible gene.

[0122] Phenotypic Analyses

[0123] Mutant phenotypes recovered using the screen of the instantinvention can be categorized into several broad categories. First, usingmorphological and histological criteria mutations that visibly perturbstructural development of the pharynx, esophagus, intestine, liver andbiliary tract are distinguished from those mutants that appear normal.The latter group is considered physiological mutants and is categorizedbased upon its handling of the panel of fluorescent lipids of theinstant invention. This group encompasses, but is not limited to,mutations affecting the intestinal epithelium, liver, vasculature andlymphatics, enteric neuromusculature, and the tissue specific regulationof PLA₂.

[0124] Embryological and transient expression assays also are importantstudies that can aid phenotypic analyses of zebrafish mutants.Unfortunately, mutations affecting development of the zebrafishdigestive organs are, in general, less easily analyzed using thesetechniques than mutations affecting early development. The shorthalf-life of injected RNA transcripts and DNA expression constructscoupled with the mosaic distribution of the micro-injected DNA limitsthe utility of transient expression assays for mutations that are notrecognizable until 4-5 dpf.

[0125] In one embodiment of the instant invention, histological analysesare performed by fixing larvae in 4% paraformaldehyde, embedding thefixed larvae in glycolmethacrylate, and followed by sectioning. Sectionsare stained using toluene blue/azure II as described and analyzed usinga Zeiss Axioplan compound microscope. When needed, selectedimmunocytochemical and molecular markers are employed to furthercategorize organ specific defects. If necessary ultrastructural studiesare performed as well. For ‘physiological’ mutations, affected larvaeare sequentially soaked in the fluorescent lipids of the instantinvention, thereby allowing a more detailed categorization.

[0126] Molecular Analyses

[0127] The molecular characterization of zebrafish mutations can beperformed using techniques widely known to those of skill in the art.These techniques include, but are not limited to, use of an everexpanding array of physical and genetic markers, several large insertgenomic DNA libraries, outstanding bioinformatics, and a successful ESTprogram.

[0128] Recent announcement of plans to physically map and sequence thezebrafish genome suggest that within 2-3 years molecularcharacterization of zebrafish mutants will be greatly simplified. One ofthe major advantages of working in zebrafish compared with othervertebrates is the potential to generate high-resolution maps of mutantloci. Since mutations can be confidently mapped to within 0.1 cM, thecritical interval surrounding a mutant locus can be narrowed to includerelatively few candidate genes. The recent identification of the genesresponsible for several zebrafish mutants demonstrates this nicely.

[0129] In one embodiment of the instant invention, the molecularcharacterization of identified mutations is accomplished by introducingmutations into a polymorphic genetic background and assigning achromosomal location using either bulk segregant analyses andpolymorphic markers from the 25 zebrafish linkage groups or using halftetrads and polymorphic centromeric markers. Concomitantly, DNA fromlarge numbers of mutant progeny from the ‘map cross’ are extracted andstored. Thereafter, the genetic map of the region surrounding the locusis refined using standard PCR based techniques and genetic markers fromexisting maps.

[0130] The closest known genetic markers flanking the mutant locus arethen used to screen large insert genomic libraries to identify moreclosely linked markers. For BAC and PAC clones this involves directsequencing, whereas for YAC clones this requires rescue and sequencingof the zebrafish genomic DNA adjacent to the YAC arms. Ultimately,flanking markers within 0.1 cM of the mutant locus are identified, and aBAC or PAC clone spanning the locus is analyzed for the responsiblegene. The latter is accomplished by direct sequencing, using the genomicinsert to probe an appropriately staged genomic library and/or exontrapping. Confirmation of mutation is accomplished via expressionanalyses and mutant rescue, using transient analyses, if possible, orvia transgenesis. Identification of tightly linked flanking markersgenerally involves a BAC, PAC, or YAC chromosomal walk, using markersmapped genetically and physically (radiation hybrid panel).

[0131] In one embodiment of the instant invention, genetic mapping isperformed using PCR based techniques. SSR polymorphisms are resolvedusing denaturing (SSR) polyacrylamide gel electrophoresis while singlebase-pair polymorphisms are resolved using Conformation Sensitive GelElectrophoresis (CSGE), a non-denaturing technique related to SSCP. DNAdetection is accomplished using a Molecular Dynamics FluorescentScanner; PCRs use Cy-5 end-labeled primers. PCR fragments generatedusing non-fluorescent primers are visualized on polyacrylamide gelsafter staining with the fluorescent intercalating dye Syto-61 (MolecularProbes). For bulk segregant analyses, separate pools of DNA from 25mutant larvae and 25 sibling WT larvae are generated and the segregationof SSR or single base pair polymorphic markers derived from ESTsanalyzed.

[0132] For physical mapping, the LN 540 (104) and Goodfellow RadiationHybrid panels are employed. Markers are mapped using a PCR basedstrategy and computer assisted analysis of amplification patterns.Screening of genomic libraries, rescue of YAC ends, generation of YAC,BAC, and PAC clones is accomplished using the commercial suppliers orpublished protocols, which are known to those of skill in the art. Allother molecular techniques outlined above employ published protocols,which also are known to those of ordinary skill in the art.

EXAMPLE 4

[0133] Fat Free Mutation

[0134] The instant invention discloses methods for screening formutations that perturb lipid processing and characterizing thosemutations, both phenotypically and molecularly. In one embodiment of theinstant invention, a classic two-generation breeding scheme (110, 111)was employed in the screen. Adult male zebrafish were mutagenized with 3mM ENU by placing them into an aqueous solution for three (3) 1 hrperiods within 1 week. At >4 weeks after the ENU treatment, the G0 maleswere bred to homozygosity using a classical 3 generation protocol (F3screen) (110, 111). Employing this approach, the segregation ofrecessive mutations was easily tested, and the F2 parents of a crossproducing mutant F3 embryos were identified as heterozygous carriers forthe mutation.

[0135] Larval progeny of individual F2 families were bathed in PED6 andscreened for digestive and organ morphology and phospholipid processing.Of the 190 genomes screened, two mutations were identified and confirmedin the subsequent generation. The recessive lethal mutation canolaproduced a phenotype closely resembling that of slj and pie, withdegeneration of the intestinal epithelium and reduced gall bladder andintestinal fluorescence. The fat free recessive lethal mutation causes amore pronounce reduction in fluorescence (FIG. 14B). However, incontrast to canola and other intestinal mutants, the fat free digestivetract has a normal appearance at 5 dpf (FIG. 14A). Therefore, fat freewould have escaped detection in a screen based solely on morphologicalcriteria.

[0136] The affect of the fat free mutation on larval digestivephysiology was determined. In order to ascertain whether swallowing wasnormal in fat free mutants, larvae 6 dpf were placed in EM containingfluorescent latex microspheres (0.0025% Fluoresbrite plain YG 2.0 μm,Polysciences Inc.) for 1 hr, washed, and imaged over 1 hour using aZeiss Axiocam 2 mounted on a Leica MZFL-III. Numbers of beads were 10±2in the WT versus 14±3 beads in mutant larvae (mean±SEM, n=9, P>0.3).Next, to determine whether the fat free hepatobiliary system wasfunctional, fat free larvae were injected (˜5 nl) with phenol red (2% inEM) dorsal to the anterior intestine and labeling of the gall bladderwas monitored (FIG. 14C). TLC of metabolites produced by fat free larvaelabeled with an unquenched BODIPY phosphatidylcholine analog (36)revealed that PLA₂ processing was intact. Extracts prepared frommutants, however, contained significantly reduced levels of fluorescentcleavage products (FIG. 14D).

[0137] Lipid processing was examined further using a fluorescentcholesterol analog (112) whose absorption is dependent on biliaryemulsification. Cholesterol was absorbed poorly by fat free larvae (FIG.14E). Uptake of BODIPY FL-C5, a more soluble short-chain fatty acid, wasreduced to a lesser extent than uptake of cholesterol, thus suggestingthat fat free directly regulates bile synthesis or secretion rather thanintestinal lipid absorption or transport. The fat free mutation providesin vivo evidence supporting the hypothesis, derived from mammalian cellculture experiments, that phospholipid turnover and cholesterolmetabolism are coupled.

[0138] The fat free fish will be useful in assessing phospholipid and/orcholesterol processing and will aid in identifying treatments fordiseases and disorders associated with phospholipid and/or cholesterolmetabolism.

[0139] Although mutant zebrafish represent a preferred embodiment of theinvention, other mutant organisms, including without limitation mutantamphibia, mutant teleosts, and mutant mammals (for example, rodents,pigs, cattle, and sheep) can be constructed by standard techniques areare included within the scope of this invention.

EXAMPLE 5

[0140] Drug Screening Assays

[0141] The invention provides methods for identifying compounds oragents that can be used to treat disorders characterized by (orassociated with) aberrant or abnormal lipid and/or cholesterolmetabolism. These methods are also referred to herein as drug screeningassays and typically include the step of screening a candidate/testcompound or agent for the ability to modulate (e.g., stimulate orinhibit) lipid and/or cholesterol metabolism. Candidate/test compoundsor agents that have one or more of these abilities can be used as drugsto treat disorders characterized by aberrant or abnormal lipid and/orcholesterol metabolism. Candidate/test compounds or agents include, forexample, (1) peptides such as soluble peptides, including Ig-tailedfusion peptides and members of random peptide libraries (see e.g., Lam,K. S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature354:84-86) and combinatorial chemistry-derived molecular libraries madeof D- and/or L-configuration amino acids; (2) phosphopeptides (e.g.,members of random and partially degenerate, directed phosphopeptidelibraries, see. e.g., Songyang, Z. et al. (1993) Cell 72:767-778); (3)antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic,chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fabexpression library fragments, and epitope-binding fragments ofantibodies); and (4) small organic and inorganic molecules (e.g.,molecules obtained from combinatorial and natural product libraries).

[0142] In one embodiment, the invention provides a method foridentifying a compound capable of use in the treatment of a disordercharacterized by (or associated with) aberrant or abnormal lipid and/orcholesterol metabolism. This method typically includes the step ofassaying the ability of the compound or agent to modulate lipid and/orcholesterol metabolism, thereby identifying a compound for treating adisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism. Disorders characterized by aberrant or abnormal lipid and/orcholesterol metabolism are described herein. The invention providesscreening assays to identify candidate/test compounds or agents thatmodulate lipid and/or cholesterol metabolism. Typically, the assaysinclude the steps of identifying at least one phenotypic perturbation oflipid and/or cholesterol metabolism in an organism, administering atleast one quenched or fluorescently-tagged phospholipid, cholesterol orother lipid analogue to the organism having the phenotypic perturbation,administering a candidate/test compound or agent to the organism underconditions that allow for the uptake of the candidate/test compound oragent by the organism and wherein but for the presence of thecandidate/test compound or agent the pattern of fluorescence would beunchanged, and detecting a change in the pattern of fluorescence bycomparing the pattern of fluorescence prior to candidate/test compoundor agent administration with that seen following administration of thecandidate/test compound or agent.

[0143] In another embodiment, a screening procedure for identifyingtherapeutic compounds that affect phospholipid and/or cholesterolmetabolism in vivo is disclosed. In general, the method involvesscreening any number of compounds for therapeutically active agents byemploying a mutant organism, for example but not limited to fish havingthe fat free mutation, described herein. Based upon the above results,it will be readily understood that a compound that affects phospholipidand/or cholesterol metabolism in an organism (e.g., the mutant fishdescribed herein) can provide an effective therapeutic agent in a mammal(e.g., a human patient). Since the screening procedures of the inventionare performed in vivo, it can be ascertained whether candidate compoundswill be highly toxic to a mammalian host organism (e.g., a humanpatient). In addition, the invention also makes available highthroughput in vitro screening methods for screening of large quantitiesof candidate compounds using the cells and cell lines of the invention.

[0144] Accordingly, the methods, materials, and organisms of the instantinvention simplify the evaluation, identification, and development ofactive agents, such as drugs, for the treatment of a variety of diseasesassociated with phospholipid and/or cholesterol metabolism. In general,the screening methods of the invention provide a facile means forselecting any number of compounds of interest from a large populationthat are further evaluated and condensed to a few active and selectivematerials. Constituents of this pool can then be evaluated in themethods of the invention to determine their activity.

[0145] Administration of the candidate compound to an organism of theinvention can be by any known route, and at a range of concentrations.Following an appropriate period of time, the animal can be assessed forthe effect of the compound compared to control animals.

[0146] Cells from the mutant organisms of the invention are also usefulas a source of cells for cell culture.

[0147] Mutagenesis screens using fluorescent lipids exploit theadvantages of combining genetic analyses with imagining of enzymaticfunction. The existence of related lipid processing mechanisms inmammals and teleosts, and the finding that therapeutic drugs used tomodify lipid metabolism in humans are active in zebrafish, establishthat genetic screens can be designed to probe the mechanistic basis ofacquired and heritable human disorders. The evidence of the instantinvention demonstrates that lipid metabolism in mammals and fish can bemonitored with fluorescent lipids, and that such organisms metabolizeingested fluorescent lipids in an analogous manner. Consequently, themethods for using the fluorescent lipids of the instant invention tostudy lipid metabolism, identify diseases of lipid metabolism, and/or toidentify agents to treat therapeutically or prophylatically diseases ordisorders of lipid metabolism and genetic screening are applicable toall vertebrate model organisms, including, but not limited to, rodents,amphibia, and fish. Fluorescent reporters are predicted to identifygenes, including but not limited to, genes involved in diseases of lipidmetabolism, such as atherosclerosis; in disorders of biliary secretion,such as biliary atresia; and in cancer, a disease in which lipidsignaling plays an important role. Identification of these genes hasimportant implications for cancer research and research related todiseases of the liver, intestine and cardiovascular system.

[0148] While this invention has been described with a reference tospecific embodiments, it will be obvious to those of ordinary skill inthe art that variations in these methods and compositions may be usedand that it is intended that the invention may be practiced otherwisethan as specifically described herein. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the claims.

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We claim:
 1. A mutant fish whose genome comprises a homozygousdisruption in a metabolic pathway of cholesterol and/or lipidprocessing, wherein said disruption reduces cholesterol absorption in agall bladder of said mutant fish and wherein said mutant fish arephenotypically normal.
 2. A mutant fish of claim 1, wherein thehomozygous disruption results in a reduction in cholesterol absorptionin a gall bladder of said mutant fish as compared to wild-type fish. 3.A method of identifying an agent(s) to treat either prophylatically ortherapeutically a disease or disorder characterized by aberrant orabnormal lipid and/or cholesterol metabolism, comprising: (a)administering a fluorescent label to said mutant fish of claim 1 byproviding at least one fluorescent lipid to said mutant fish underconditions that allow for uptake of said fluorescent lipid by saidmutant fish; (b) selecting at least one agent to treat a disease ordisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism; (c) administering said agent(s) to said mutant fish underconditions that allow for uptake of said agent(s) by said mutant fishprior to, after, or simultaneously with step (a); (d) analyzing a changein the pattern and/or rate of fluorescence in said mutant fish as anindication of the efficacy of said agent(s) to treat a disease ordisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism; and (e) identifying an agent(s) to treat eitherprophylatically or therapeutically a disease or disorder characterizedby aberrant or abnormal lipid and/or cholesterol metabolism by a changein the pattern and/or rate of fluorescence in said mutant fish.
 4. Amethod of identifying an agent(s) to reduce lipid and/or cholesterolmetabolism, comprising: (a) administering a fluorescent label to saidmutant fish of claim 1 by providing at least one fluorescent lipid tosaid mutant fish under conditions that allow for uptake of saidfluorescent lipid by said mutant fish; (b) selecting at least one agentto treat a disease or disorder characterized by aberrant or abnormallipid and/or cholesterol metabolism; (c) administering said agent(s) tosaid mutant fish under conditions that allow for uptake of said agent(s)by said mutant fish prior to, after, or simultaneously with step (a);(d) analyzing a change in the pattern and/or rate of fluorescence insaid mutant fish as an indication of the efficacy of said agent(s) totreat a disease or disorder characterized by aberrant or abnormal lipidand/or cholesterol metabolism; and (e) identifying an agent(s) to treateither prophylatically or therapeutically a disease or disordercharacterized by aberrant or abnormal lipid and/or cholesterolmetabolism by a change in the pattern and/or rate of fluorescence insaid mutant fish.
 5. A method of identifying an agent(s) to enhancelipid and/or cholesterol metabolism, comprising: (a) administering afluorescent label to said mutant fish of claim 1 by providing at leastone fluorescent lipid to said mutant fish under conditions that allowfor uptake of said fluorescent lipid by said mutant fish; (b) selectingat least one agent to treat a disease or disorder characterized byaberrant or abnormal lipid and/or cholesterol metabolism; (c)administering said agent(s) to said mutant fish under conditions thatallow for uptake of said agent(s) by said mutant fish prior to, after,or simultaneously with step (a); (d) analyzing a change in the patternand/or rate of fluorescence in said mutant fish as an indication of theefficacy of said agent(s) to treat a disease or disorder characterizedby aberrant or abnormal lipid and/or cholesterol metabolism; and (e)identifying an agent(s) to treat either prophylatically ortherapeutically a disease or disorder characterized by aberrant orabnormal lipid and/or cholesterol metabolism by a change in the patternand/or rate of fluorescence in said mutant fish.
 6. A method ofidentifying an agent(s) to reduce cholesterol absorption, comprising (a)administering a fluorescent label to said mutant fish of claim 1 byproviding at least one fluorescent lipid to said mutant fish underconditions that allow for uptake of said fluorescent lipid by saidmutant fish such that fluorescence is observed in a gall bladder of saidmutant fish; (b) selecting at least one agent to reduce cholesterolabsorption in said mutant fish; (c) administering said agent(s) to saidmutant fish under condition that allow for uptake of said agent(s) bysaid mutant fish prior to, after, or simultaneously with step (a); (d)analyzing a change in the pattern and/or rate of fluorescence in saidmutant fish as an indication of the efficacy of said agent(s) to reducecholesterol absorption; and (e) identifying an agent(s) to reducecholesterol absorption by a reduction in the pattern and/or rate offluorescence in a gall bladder of said mutant fish.
 7. A method ofidentifying candidate functional mediators of the homozygous disruptionin the mutant fish of claim 1, comprising (a) producing an F₁ generationfish by crossing a first mutant fish whose genome comprises aheterozygous disruption in the metabolic pathway of cholesterol and/orlipid processing, wherein said disruption when homozygous reducescholesterol absorption in a gall bladder of a fish having saidhomozygous disruption but produces no phenotypic change in a fish havingsaid homozygous disruption, with a second mutant fish whose genomecomprises at least one random mutation; (b) selecting from the offspringof the cross of step (a) those fish whose genome comprises theheterozygous disruption of said first mutant fish; (c) producing an F₂generation fish by intercrossing the offspring selected in step (b); (d)selecting from the offspring of the cross of step (c) those fish whosegenome comprises the heterozygous disruption of the mutant fish of claim1; (e) producing an F₃ generation fish by intercrossing the offspringselected in step (d); (f) administering a fluorescent label to theselected F₃ offspring of step (d) by providing at least one quenchedfluorescent phospholipid analogue and/or NBD cholesterol to saidselected F₃ offspring under conditions that allow for uptake of saidquenched fluorescent phospholipid and/or NBD cholesterol by saidselected F₃ offspring; (g) comparing the pattern and/or rate offluorescence in said selected F₃ offspring to the pattern and/or rate offluorescence observed in a wild-type fish and/or mutant fish of claim 1,which wild-type fish and/or mutant fish of claim 1 was administered afluorescent label by providing at least one quenched fluorescentphospholipid analogue and/or NBD cholesterol to said wild-type fishand/or mutant fish under conditions that allow for uptake of saidquenched fluorescent phospholipid and/or NBD cholesterol by saidwild-type fish and/or mutant fish of claim 1; and (h) identifyingcandidate functional mediators of the homozygous disruption in themutant fish of claim 1 by a change in the pattern and/or rate offluorescence in said selected F₃ offspring as compared to the patternand/or rate of fluorescence in wild-type fish and/or mutant fish ofclaim
 1. 8. A cell line derived from the mutant fish of claim
 1. 9. Thecell line of claim 8, wherein the cell line is derived from a liver ofthe mutant fish of claim
 1. 10. The cell of claim 8, wherein the cellline is derived from a gall bladder of the mutant fish of claim
 1. 11. Amethod of identifying an agent(s) to treat either prophylatically ortherapeutically a disease or disorder characterized by aberrant orabnormal lipid and/or cholesterol metabolism, comprising: (a)fluorescently labeling a cell isolated from at least one of the celllines of claim 8, 9, or 10 by contacting said cell with at least onefluorescent lipid under conditions that allow for uptake of saidfluorescent lipid by said cell; (b) selecting at least one agent totreat a disease or disorder characterized by aberrant or abnormal lipidand/or cholesterol metabolism; (c) contacting said agent(s) with saidcell under conditions that allow for uptake of said agent(s) by saidcell prior to, after, or simultaneously with step (a); (d) analyzing achange in the pattern and/or rate of fluorescence in said cell as anindication of the efficacy of said agent(s) to treat a disease ordisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism; and (e) identifying an agent(s) to treat eitherprophylatically or therapeutically a disease or disorder characterizedby aberrant or abnormal lipid and/or cholesterol metabolism by a changein the pattern and/or rate of fluorescence in said cell.
 12. A method ofidentifying an agent(s) to reduce lipid and/or cholesterol metabolism,comprising: (a) fluorescently labeling a cell isolated from at least oneof the cell lines of claim 8, 9, or 10 by contacting said cell with atleast one fluorescent lipid under conditions that allow for uptake ofsaid fluorescent lipid by said cell; (b) selecting at least one agent totreat a disease or disorder characterized by aberrant or abnormal lipidand/or cholesterol metabolism; (c) contacting said agent(s) with saidcell under conditions that allow for uptake of said agent(s) by saidcell prior to, after, or simultaneously with step (a); (d) analyzing achange in the pattern and/or rate of fluorescence in said cell as anindication of the efficacy of said agent(s) to treat a disease ordisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism; and (e) identifying an agent(s) to treat eitherprophylatically or therapeutically a disease or disorder characterizedby aberrant or abnormal lipid and/or cholesterol metabolism by a changein the pattern and/or rate of fluorescence in said cell.
 13. A method ofidentifying an agent(s) to enhance lipid and/or cholesterol metabolism,comprising: (a) fluorescently labeling a cell isolated from at least oneof the cell lines of claim 8, 9, or 10 by contacting said cell with atleast one fluorescent lipid under conditions that allow for uptake ofsaid fluorescent lipid by said cell; (b) selecting at least one agent totreat a disease or disorder characterized by aberrant or abnormal lipidand/or cholesterol metabolism; (c) contacting said agent(s) with saidcell under conditions that allow for uptake of said agent(s) by saidcell prior to, after, or simultaneously with step (a); (d) analyzing achange in the pattern and/or rate of fluorescence in said cell as anindication of the efficacy of said agent(s) to treat a disease ordisorder characterized by aberrant or abnormal lipid and/or cholesterolmetabolism; and (e) identifying an agent(s) to treat eitherprophylatically or therapeutically a disease or disorder characterizedby aberrant or abnormal lipid and/or cholesterol metabolism by a changein the pattern and/or rate of fluorescence in said cell.
 14. A method ofidentifying candidate functional mediators of the homozygous disruptionin the mutant fish of claim 1, comprising (a) providing a firstfluorescent label with a cell isolated from at least one of the celllines of claim 8, 9, or 10 by contacting at least one quenchedfluorescent phospholipid analogue and/or NBD cholesterol with said cellunder conditions that allow for uptake of said quenched fluorescentphospholipid and/or NBD cholesterol to said cell; (b) analyzing thepattern and/or rate of fluorescence of said first fluorescent label insaid cell; (c) randomly re-mutagenizing the DNA of said cell; (d)providing a second fluorescent label to said cell by contacting at leastone quenched fluorescent phospholipid analogue and/or NBD cholesterol,wherein said second fluorescent label has a fluorescence emissionprofile that contrasts with the fluorescence emission profile of saidfirst fluorescent label, with said cell under conditions that allow foruptake of said quenched fluorescent phospholipid and/or NBD cholesterol;(e) comparing the pattern and/or rate of fluorescence of said secondfluorescent label in said cell to the pattern and/or rate offluorescence observed in said cell in step (b); and (f) identifyingcandidate functional mediators of the homozygous disruption in themutant fish of claim 1 by a change in the pattern and/or rate offluorescence in said cell.